Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP-1

• Published in: Biochemical journal 2004-03, Vol.378 (Pt 3), p.919-928
• Main Authors: Qadri, Ishtiaq, Iwahashi, Mieko, Capasso, Juan M, Hopken, Matthew W, Flores, Sonia, Schaack, Jerome, Simon, Francis R
• Format: Article
• Language: English
• Subjects: Adenovirus; Cell Line; Enzyme Activation; Enzyme Inhibitors - pharmacology; Gene Expression Regulation, Enzymologic; Hepacivirus - genetics; Hepacivirus - pathogenicity; Hepacivirus - physiology; Hepatitis C virus; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases - antagonists & inhibitors; Mitogen-Activated Protein Kinases - physiology; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun - biosynthesis; Replicon; Superoxide Dismutase - genetics; Superoxide Dismutase - metabolism; Transcription Factor AP-1 - physiology; Transcriptional Activation; Viral Nonstructural Proteins - metabolism; Virus Replication
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj20031587

Activation of cellular kinases and transcription factors mediates the early phase of the cellular response to chemically or biologically induced stress. In the present study we investigated the oxidant/antioxidant balance in Huh-7 cells expressing the HCV (hepatitis C virus) subgenomic replicon, and observed a 5-fold increase in oxidative stress during HCV replication. We used MnSOD (manganese-superoxide dismutase) as an indicator of the cellular antioxidant response, and found that its activity, protein levels and promoter activity were significantly increased, whereas Cu/ZnSOD was not affected. The oxidative stress-induced protein kinases p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) were activated in the HCV repliconcontaining cells and in Huh-7 cells transduced with Ad-NS5A [a recombinant adenovirus encoding NS5A (non-structural protein 5A)], coupled with a 4-5-fold increase in AP-1 (activator protein-1) DNA binding. Ava.1 cells, which encode a replication-defective HCV replicon, showed no significant changes in MnSOD, p38 MAPK or JNK activity. The AP-1 inhibitors dithiothreitol and N -acetylcysteine, as well as a dominant negative AP-1 mutant, significantly reduced AP-1 activation, demonstrating that this activation is oxidative stress-related. Exogenous NS5A had no effect on AP-1 activation in vitro, suggesting that NS5A acts at the upstream targets of AP-1 involving p38 MAPK and JNK signalling cascades. AP-1-dependent gene expression was increased in HCV subgenomic replicon-expressing Huh-7 cells. MnSOD activation was blocked by inhibitors of JNK (JNKI1) and p38 MAPK (SB203580), but not by an ERK (extracellular-signal-regulated kinase) inhibitor (U0126), in HCV-replicating and Ad-NS5A-transduced cells. Our results demonstrate that cellular responses to oxidative stress in HCV subgenomic replicon-expressing and Ad-NS5A-transduced cells are regulated by two distinct signalling pathways involving p38 MAPK and JNK via AP-1 that is linked to increased oxidative stress and therefore to an increased antioxidant MnSOD response.